Dyes are already used as controls for assay set-ups that require pipetting of several components. These control concepts are based on dyes only within one component and detecting the secondary colour after mixture with other components.
Especially real-time PCR systems require an exact mixture of all reaction components for reliable and comparable quantification, because the amount of sample material, of primers/probes as well as of master mixes required for real time PCR particularly influence the quantification result.
PCR master mix with an inert dye is used to minimize pipetting errors and commercial products are available, such as RedTaq of Sigma Aldrich or AbsoluteBlue of ThermoScientific (Yanek, M., BIOspektrum 13(5) (2007) 519-520). Alternatively, the ingredients (such as, e.g., the polymerase) of the components may be labeled prior to mixing to obtain a visual control of the pipetting process. E.g., the RedTaq of Sigma Aldrich comprises a labeled polymerase. The U.S. Pat. No. 5,861,251 discloses lyophilized PCR reagents comprising a dye. Moreover, also the probe materials may be provided with a dye for visualization (WO 2000/014505). Fluorescent dyes such as ROX and ROX/FAM FRET pairs are used as calibration standards (U.S. Pat. No. 5,736,333).
Currently, there are no control systems known in the state of the art that provide a volume control for more than one component to be mixed for a certain assay. Especially for real-time PCR systems the control dye must be optimized such that the detection dye of the real-time PCR is not affected and consequently, the adsorption of the control dye should not overlap with the detection channels used for monitoring the PCR and the control dye should be detected in a separate channel. This becomes more difficult, if two or more control dyes should be used, because the spectral window for PCR monitoring is reduced in order to avoid cross talk with respect to all control dyes.
The present invention provides a closed system for assay set-up that encompasses control strategies to identify volume errors of at least two components to be mixed for said assays.